Method for isolating satellite sequences

ABSTRACT

A highly homogeneous library can be obtained by cleaving a genomic DNA by a sequence-independent cleavage method, such as sonication. By selecting satellite sequences from such a library, efficiency of isolation is improved. Thus, an efficient method of isolating microsatellite sequences, which are useful as DNA markers, is provided.

TECHNICAL FIELD

The present invention relates to a method for isolating satellitesequences. Satellite sequences, which are useful as population geneticmarkers, can be used as markers for genetic linkage analyses, etc.Therefore, an efficient technique for isolating them is an importantsubject of research in the field of genome analysis.

BACKGROUND ART

It has been known that eukaryotic genomes contain repetitive sequencesconsisting of similar nucleotide sequence repeats. The first discoveredrepetitive sequence had a repeat unit consisting of satellite DNA, whichis a long sequence ranging from several hundred to several thousandbases, and is called a satellite sequence (Bioscience, 27: 790-796,1977). Repetitive sequences consisting of shorter nucleotide sequenceunits were later identified. They have been named, depending on the sizeof their repeat unit, microsatellite sequences (Am. J. Hum. Genet., 4:397-401, 1989) when they have a repeat unit of 2 to 5 bases (NucleicAcids Res. 9: 5931-5947, 1981) or minisatellite sequences (Nature, 314:67-73, 1985) when they have a repeat unit of 10 to 64 bases (Nature,295: 31-35, 1982). Microsatellite sequences are also called simplesequences (Nucleic Acids Res. 17: 6463-6471, 1989) or short tandemrepeats (Am. J. Hum. Genet. 49: 746-756), etc.

Use of microsatellite sequences as markers was not reported when theywere discovered. However, after their polymorphism was confirmed bypolymerase chain reaction (PCR) (Am. J. Hum. Genet. 4: 397-401, 1989),they have attracted the attention of researchers for their use asmarkers in a variety of areas. Specifically, their application topedigree or lineage discrimination and individual identification ofhumans, animals, and plants is known. Because microsatellite sequencesare scattered throughout a genome and abound in variation, they are goodgenetic markers. Furthermore, a microsatellite DNA polymorphism containsmany polymorphic gene loci and a large number of alleles per gene locus.In addition, based on PCR, microsatellite DNA polymorphism analysis iseasy to perform. The amplified products are easily detected as single ordouble bands by electrophoresis, which simplifies determination ofindividual types and facilitates the data processing. Therefore,microsatellite DNA markers have become widely used as the most effectivemarkers for population genetics (J. Fish. Biol., 47: 29-55, 1995).

For microsatellite DNA polymorphism analysis, first, a variety ofmicrosatellite DNA should be isolated from individual species to beanalyzed. In addition, the microsatellite DNA regions should beamplified by PCR to detect the microsatellite polymorphism. Appropriateprimers are needed for PCR. This means that, to perform microsatelliteDNA polymorphism analysis, efficient methods for isolatingmicrosatellite DNA from the species and for designing PCR primerscapable of amplifying the microsatellite region are required.

The inventors have already reported a method that is expected to allowefficient isolation of microsatellites from poultry, for whichmicrosatellite polymorphism analysis is not advanced (Jpn. Poult. Sci.,33: 292-299, 1996). This method is an improvement on a conventionalmethod of microsatellite sequence isolation that consists of a series ofmanipulations: fragmentation of a genome with restriction enzymes,insertion of the fragments into a vector, extension reaction with(TG)_(n) primers, and then cloning (Proc. Natl. Acad. Sci. USA 89:3419-3423, 1992). Namely, isolation of unknown microsatellite sequenceswas achieved by choosing a vector with high transformation efficiencies,by specifically digesting single-stranded DNA with mung bean nuclease,or by removing bacterial DNA and RNA with DNase I and RNase A. Inchicken, it is considered difficult to efficiently isolatemicrosatellite sequences, because the number of microsatellite sequencesis small. The above method could achieve, by calculation, six times moreefficient isolation than the known method that was used in an attempt toisolate chicken microsatellite sequences (Poultry Science, 74:1855-1874, 1995).

However, even by this method, it was impossible to prevent problematicclones from contaminating the microsatellite DNA clones obtained.Namely, a high rate of duplicate clones was found among the clonesobtained, suggesting bias in the constitution of clones as well as lowefficiency.

Microsatellite sequences are required to be isolated according to eachspecies. There are few species for which the isolation of microsatellitesequences is in progress, and it is still necessary to isolatemicrosatellile sequences from many species. However, there are a numberof species-specific problems, for example, low frequency ofmicrosatellite sequences in the chicken genome. Therefore, it is usefulto provide a novel technique for isolating microsatellite sequences,also because it will enable selection from a variety of approaches.

DISCLOSURE OF THE INVENTION

An objective of the present invention is to provide a method forisolating satellite sequences with high isolation efficiency. Thepresent invention provides an isolation method more efficient than anyknown technique.

The present inventors presumed that one of the major causes forgenerating duplicate clones during isolation of satellite sequences byknown techniques might come from the fragmentation of the genomic DNA.In other words, the possibility that, when digested with restrictionenzymes, the genomic library is biased due to the fact thatsequence-specific cleavage cannot be excluded.

Therefore, the inventors attempted to utilize a nucleotidesequence-independent cleavage method in which more arbitrary cleavage ofthe genomic DNA is expected. As a result, they found that cleavage ofgenomic DNA is possible with nucleotide sequence-independentDNA-digesting enzymes or by a physical action. However, since most ofthe physically cleaved genomic DNA fragments are not phosphorylated attheir ends, they are not allowed to be efficiently incorporated intovectors using an enzyme. In addition, since physically cleaved fragmentshave an irregularly protruding strand at their cleavage site, theycannot be ligated. The inventors have overcome these problems by usingseveral enzymatic treatments, and have established a method to obtain amore homogeneous genomic library. Furthermore, they have confirmed thatthe library thus obtained enables efficient isolation of satellitesequences, thereby completing the present invention. As used herein, theterm “satellite sequences” indicates, unless otherwise mentioned, anyrepeat sequences which include microsatellite sequences andminisatellite sequences. Also as used herein, “microsatellite sequences”and “minisatellite sequences” indicate repeat sequences consisting ofrepeat units of 2 to 5 bp and 10 to 64 bp, respectively. Thus, thepresent invention relates to the following methods for isolatingsatellite sequences.

-   (1) An isolation method for satellite sequences, wherein a genomic    DNA is cleaved by a nucleotide sequence-independent method, the    isolation method comprising:-   a) obtaining randomly cleaved fragments of the genomic DNA and-   b) selecting, from the fragments obtained in a), fragments    comprising the satellite sequences.-   (2) The isolation method of (1), wherein the nucleotide    sequence-independent method is a physical cleavage method or an    enzymatic cleavage method.-   (3) The isolation method of (2), wherein the physical cleavage    method is sonication.-   (4) The isolation method of (3), wherein the ends of the genomic DNA    that have been fragmented by sonication are to be blunted.-   (5) The isolation method of (4), wherein the ends are to be blunted    with DNA polymerase having single strand-specific endonuclease    activity and 3′→5′ exonuclease activity.-   (6) The isolation method of (2), wherein a nucleotide    sequence-independent endonuclease is used in the enzymatic cleavage    method.-   (7) The isolation method of (6), wherein the nucleotide    sequence-independent endonuclease is DNase I.-   (8) The isolation method of (1), wherein the satellite sequences are    microsatellite sequences.-   (9) Use of satellite sequences isolated by the isolation method of    any one of (1) to (8) as DNA markers.

In the present invention, it is critical to cleave a genomic DNA using anucleotide sequence-independent cleavage method that gives randomfragments. Cleavage of a genomic DNA using a nucleotidesequence-independent cleavage method refers to cleaving DNA withoutdependence on structural characteristics (i.e., nucleotide sequences) ofthe DNA. Therefore, neither chemical reactions in which DNA is cleavedin a nucleotide sequence-dependent manner nor digestion with restrictionenzymes constitutes the cleavage method of the present invention. Thegenomic DNA to which the method of the present invention is to beapplied can be prepared by known methods. Namely, cells are lysedenzymatically with protease, and are subjected to DNA extraction usingan appropriate solvent.

For cleavage of a genomic DNA using a nucleotide sequence-independentcleavage method, any of the above-described cleavage methods, forexample, those based on physical actions and those based on the actionof DNA-digesting enzymes that do not recognize the nucleotide sequence,can be used. Physical actions include sonication and agitation. On theother hand, as DNA-digesting enzymes that do not recognize thenucleotide sequence, DNase I and the like are known. Any of thesemethods can be used in the present invention. Above all, treatment bysonication is one of the most desirable manipulations with excellentreproducibility. By sonication, a large quantity of fragments withuniform lengths can be obtained in a short time. In contrast, partialdigestion of a genomic DNA with DNase I requires strict setting ofconditions for reproducibly obtaining fragments with uniform lengths. Inaddition, this method often requires troublesome manipulations.

As to the extent of cleavage of a genomic DNA, those skilled in the artcan empirically set the conditions that will permit efficient productionof fragments having the optimal size for the satellite sequence ofinterest. For example, if isolation of microsatellite sequences isintended, such conditions that give a library of fragments of 300 to 900bp are selected. Considering the operation of ligating the cleavedfragments into a vector, it is desirable to use conditions under whichthe majority of fragments generated have a size of 300 to 500 bp or 500to 800 bp. This is because, if fragments contain integral multiples ofsmaller fragments, such fragments are indistinguishable from the ligatedproducts of more than one smaller fragment. For instance, whensonication at 20 kHz with an amplitude of 10 is performed on ice,fragments of 300 to 500 bp can be efficiently obtained if a sonicationof 1-minute duration is performed 1 to 5 times. Since a prolongedsonication may heat the sample and denature DNA, it is desirable torepeat the sonication with a shorter time duration.

The genomic DNA cleaved by physical actions such as sonication tends toexhibit poor ligation efficiency, which prevents efficient incorporationof the DNA into vectors. For this reason, it is desirable to blunt theends of the DNA fragments. In the present invention, any of the knownblunting methods can be used: removal of the protruding ends with singlestrand-specific endonuclease, and further synthesis of the complementarystrand at the 3′ recessed ends with DNA polymerase to ensure theblunting. Specifically, mung bean nuclease and S1 nuclease can be usedas the single strand-specific endonucleases. On the other hand, DNApolymerase having 3′→5′ exonuclease activity can be used as the DNApolymerase. Such DNA polymerases include T4 DNA polymerase, pfupolymerase, and KOD polymerase. Mung bean nuclease, which is used in theExample, is a single strand-specific endonuclease and achieves bluntingby digesting the protruding portion of the single strands of the DNA. Inthe Example, the action of T4 DNA polymerase is further utilized toensure the blunting. T4 DNA polymerase digests the protrusion at the 3′ends by its strong 3-→5′ exonuclease activity, and at the same time, itcan synthesize complementary strands at the 3′ recessed ends, therebygiving blunt ends. However, T4 DNA polymerase cannot blunt a recessed 3′end with a phosphate group. Therefore, it is desirable to use mung beannuclease and T4 DNA polymerase together. In such a case, treatment withmung bean nuclease cleaves gaps generated in the double-stranded DNA bysonication, as well as blunts the ends. As a result, fragments withlengths shorter than expected may be produced. In order to obtain alibrary free from shorter fragments, it is desirable to provide a stepof isolating the fragments of expected size after treating with mungbean nuclease. Fragments of expected sizes can be isolated by agarosegel electrophoresis or by gel filtration. The blunt-ended genomic DNAfragments can be directly ligated into vectors. Alternatively, they canbe phosphorylated at their 5′ ends to improve the efficiency ofligation. T4 polynucleotide kinase can be used for phosphorylation.

The genomic DNA fragments with phosphorylated 5′ ends can self-ligateduring the ligation into the vector. To prevent the self-ligation, theconcentration of the fragments should be reduced, while excessconcentration of the vector is employed so as to increase theopportunities for the fragments to be ligated into the vector. For theligation reaction, T4 DNA ligase works in the ligation buffer andligation efficiency can be further improved by carrying out the ligationin the presence of the restriction enzyme used to cleave the vector. Forexample, when pCR-ScriptSK(+) is used as the vector, restriction enzymeSrfI can help keep the vector in a linear state. Since SrfI does not acton the vector into which an insert is ligated, one can expect theligation efficiency to be improved.

In the present invention, not only physical actions but also nucleotidesequence-independent DNA-digesting enzymes can be used for fragmentationof DNA. These enzymes include, for example, DNase I, etc. For enzymatictreatment with DNase I, conditions that allow uniform action of theenzyme on the whole genome and that also allow production of fragmentswith expected sizes should be empirically provided. For example, inorder to maintain the enzymatic action in a uniform state, genomic DNAwith highest purity should be used after nuclear proteins are removed.Also, the enzymatic reaction should be performed as quickly as possiblein order to avoid producing a large number of small fragments. Inaddition, conditions such as reaction time and temperature should beaccurately controlled in order to maintain high reproducibility. Afterthe enzymatic reaction is completed, the reaction is stopped by heatingor other means. The nucleic acid component is recovered from thereaction, and, if necessary, particular fragments are extracted,followed by incorporation of the fragments. into the vectors asdescribed above. The fragments of the genomic DNA digested with theenzyme are blunt-ended and phosphorylated, which allows them to bedirectly used for ligation.

The vectors used in the present invention are not specially restricted.Specifically, known vectors such as pCR-ScriptSK(+), pBluescriptKS(+)(both are manufactured by Stratagene), and pUC18 can be used. After anappropriate host is transformed with the vector including the insert andis grown, the DNA is recovered to make a genomic library. WhenpCR-ScriptSK(+) is used for transformation, it may be advantageous touse host strains having excellent transformation efficiencies, such asE. coli (competent cell) XL1-Blue MRF′, XL2-Blue MRF′, and TG1 (all aremanufactured by Stratagene).

Using the genomic library thus constructed, transformants containing thesatellite sequences of interest can be cloned. Screening for thesatellite sequences can be performed based on colony hybridization usinga probe for a satellite sequence (Can. J. Fish. Biol. 51: 1959-1996,1994) or on primer extension (Proc. Natl. Acad. Sci. USA, 89: 3419-3423,1992). In colony hybridization, colonies of the transformed cells asdescribed above are transferred onto a filter, and hybridized with aprobe having, for example, a repeat sequence of (GT)_(n). The nucleotidesequence of the oligonucleotide that constitutes the probe is properlyprovided based on the satellite sequence of interest. Positive coloniesare separated to isolate the clones containing the satellite sequence.

In primer extension, on the other hand, the genomic library is recoveredas single-stranded DNA. For example, if pCR-ScriptSK(+) is used as thevector, the transformed E. coli cells can be infected with helper phageVCS-M13 or the like to recover the inserts in a single-stranded form asrecombinant phages. Since DNA packaged in the phage is protected againstenzymatic action, RNA and DNA derived from E. coli can be enzymaticallydegraded when the DNA of interest is recovered, thereby achieving moreefficient cloning.

To the single-stranded plasmid DNA isolated from the phage, a primerspecific to the satellite sequence is annealed, and then, DNA polymeraseis applied. For example, repetitive sequences of (dA-dT)_(n) aregenerally (0.3% of the entire genomic DNA) found in the mammalianmicrosatellite sequences. In humans, such combinations as(dC-dA/dT-dG)_(n) and (dC-dT/dA-dG)_(n) are also frequently observed.Based on such information, one can design primers that comprise anucleotide sequence complementary to a satellite sequence and thatanneal to the satellite sequence. It is advantageous to phosphorylatethe 5′ end of the primer beforehand. Vectors that contain amicrosatellite sequence acquire the primer at this site and adouble-stranded DNA is synthesized, while those to which the primer doesnot anneal remain single-stranded. Ligation is performed after thesynthesis of the complementary strand with DNA polymerase to completethe double-stranded circular DNA, and then the single-stranded DNA isdigested with mung bean nuclease or the like. Consequently, only thevectors containing the satellite sequences remain. These vectors can berecovered and used to transform cells, thereby cloning the vectors.After amplifying the cloned vectors containing the satellite sequences,one can recover the DNA and determine the sequences, thereby completingthe isolation of the satellite sequences.

When a satellite sequence is isolated together with its flankingregions, primers for PCR can be designed. For designing the primers, itis convenient to utilize a commercially available package of softwarefor sequence analysis. For example, in the Example below, a primerdesigning software Primer Premier (Premier Biosoft International) isused. The microsatellite sequences and minisatellite sequences isolatedare useful as fingerprint markers for intraspecies analysis or aspedigree (or lineage) markers. If any satellite sequence obtained in thepresent invention exhibits a polymorphism, such a nucleotide sequencecan be used as an indicator for identification of individuals. Such asatellite sequence is called a fingerprint marker. Since satellitesequences used as fingerprint markers are inherited from the parents ofan individual, analysis of the fingerprint markers conserved amongmultiple individuals indicates the probability that these individualsbelong to a single pedigree. Such use of a satellite sequence is calleda pedigree (or lineage) marker.

Furthermore, the satellite sequences isolated in the present inventioncan also be used as genetic linkage markers, in addition to the use asfingerprint markers and pedigree (or lineage) markers. In the presentinvention, a satellite marker utilized as an indicator represented bythe uses exemplified above is called a DNA marker.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the image of a dot-blot assay with a microsatellitesequence-specific probe ((CA)₁₀ oligonucleotide).

BEST MODE FOR CARRYING OUT THE INVENTION

The present invention is described more specifically in the followingexamples.

(1) Preparation of DNA Fragments from Black Abalone

Black abalone (Haliotis discus discus) DNA was isolated from foot muscleaccording to the TNES-Urea method (Fisheries Sci., 62: 723-726, 1996).However, 4 M urea was used herein. The genomic DNA solution (20 μg/ml,550 μl) was sonicated (20 kHz, Amplitude 10, for one minute, five times)with cooling to fragment the DNA. A reaction mixture (52 μl) containingthe substrate DNA (about 10 μg), 30 mM CH₃COONa (pH 4.6), 50 mM NaCl, 1mM (CH₃COO)₂Zn, 5% glycerol, and 60 units of mung bean nuclease (Toyobo)was incubated for 1 hour at 37° C. to cleave the gap portions of the DNAfragments and to blunt the protruding ends. By electrophoresis on a 1.2%agarose gel, DNA fragments of 300 to 500 bp were recovered. A reactionmixture (100 μl) containing the recovered substrate DNA, 20 μM dNTP, 50mM Tris/HCl (pH 8.5), 7 mM MgCl₂, 15 mM (NH₄)₂SO₄, 10 mM2-mercaptoethanol, 0.1 mM EDTA, and 10 units of T4 DNA polymerase(Toyobo) was incubated for 1 hour at 37° C. to blunt the protruding endsof the DNA fragments. Further, a reaction mixture (50 μl) containing thesubstrate DNA, 0.2 mM rATP, 50 mM Tris/HCl (pH 7.6), 10 mM MgCl₂, 10 mM2-mercaptoethanol, and 10 units of T4 polynucleotide kinase (Toyobo) wasincubated for 1 hour at 37° C. to phosphorylate the 5′ ends of the DNAfragments.

(2) Ligation of Genomic DNA Fragments into Plasmid Vectors

A reaction mixture (10 μl) containing pCR-Script SK(+) vector(Stratagene) (about 1 μl), 25 mM Tris/Acetate (pH 7.6), 100 mM KOAc, 10mM MgOAc, 0.5 mM 2-mercaptoethanol, 10 μl/ml BSA, and 10 units ofrestriction enzyme SrfI (Stratagene) was incubated for 1 hour or more at37° C., to cleave the vector at the SrfI site. A reaction mixture (50μl) containing the vector solution prepared, the substrate DNA (about 3μg), 10 units of SrfI, 0.5 mM rATP, 66 mM Tris/HCl (pH 7.6), 6.6 mMMgCl₂, 10 mM dithiothreitol, and 20 units of T4 DNA ligase (Toyobo) wasincubated overnight at 24° C. to ligate the DNA fragments into thevector. After inactivating the DNA ligase by heating at 65° C. for 15minutes, 10 units of SrfI was further added. The reaction mixture wasincubated for 1 hour at 37° C. to digest the self-ligated vector.

(3) Preparation of Single-Stranded DNA

To each of five culturing tubes, 100 μl of XL2-Blue MRF′ ultracompetentcells (Epicurian coli ultracompetent cells; Stratagene) was added, andthen, the ligated vector (about 200 ng) was added, followed bytransformation according to the attached instruction manual. After 900μl of NZY medium (Maniatis, Molecular Cloning: A Laboratory Manual, 2nded., Cold Spring Harbor Laboratory Press, New York, 1989) was added toeach of the tubes, the cells were cultured for one hour at 37° C. withshaking. Subsequently, ampicillin was added to a final concentration of50 μg/ml. The cells were cultured for another hour at 37° C. withshaking to select the transformed E. coli. To each of the tubes, about10¹⁰ pfu of VCS-M13 helper phage was added. The cells were allowed tostand for 20 minutes at 37° C., followed by addition of kanamycin at afinal concentration of 70 μg/ml. The cells were cultured for 1 hour at37° C. with shaking to select the E. coli infected with the helperphage. All the cultures were put together and added to 100 ml ofTerrific medium (Maniatis, Molecular Cloning: A Laboratory Manual, 2nded., Cold Spring Harbor Laboratory Press, New York, 1989) containingampicillin and kanamycin. The cells were cultured for 14 hours at 37° C.with shaking. The Terrific medium was centrifuged at 14,000 rpm for 10minutes at 0° C., and the supernatant was recovered and filtered twicethrough a 45 μm filter. After precipitating twice with PEG according tothe standard method (Muramatsu, Lab Manual of Genetic Engineering, 3rded., Maruzen, Tokyo, 1996, pp.51-55), the DNA and RNA derived from E.coli were digested according to a known method (Jpn. Poult. Sci. 33:292-299, 1996). Subsequently, PEG precipitation, phenol extraction, andethanol precipitation were performed to purify the single-stranded DNA.

(4) Selection of (CA)_(n)-Positive Plasmids

Primer extension was performed using a (CA)₁₂ oligonucleotide to selectthe plasmid DNA into which DNA fragments with (TG/CA) repeats had beeninserted. The solution (98 μl) containing the single-stranded DNA (about3 μg), 0.2 mM dNTP, 20 mM Tris/HCl (pH 8.8), 10 mM KCl, 10 mM )₂SO₄, 2mM MgSO₄, 100 μg/ml BSA, 0.1% Triton X-100, and 100 pmol (CA)₁₂oligonucleotide was pre-heated at 72° C. for 10 minutes. Five units ofPfu DNA polymerase (Stratagene) was added to the solution, which wasthen overlaid with mineral oil and incubated for 30 minutes at 72° C.Subsequently, the generated double-stranded DNA was recovered by phenolextraction and ethanol precipitation and was dissolved in 10 μl ofsterilized water. After circularizing the double-stranded DNA by using aligation kit (Ligation high, Toyobo), the DNA ligase was inactivated byheat treatment (65° C. for 15 minutes). A reaction mixture (100 μl)containing this solution, 30 mM CH₃COONa (pH 4.6), 50 mM NaCl, 1 mM(CH₃COO)₂Zn, 5% glycerol, and 30 units of mung bean nuclease (Toyobo)was incubated for 2 hours or more at 37° C. to digest thesingle-stranded DNA which had not been primer-extended. DNA wasrecovered by phenol/chloroform extraction and ethanol precipitation, andwas dissolved in 60 μl of TE buffer.

With the recovered DNA (equivalent to 100 μg of the single-strandedDNA), XL2-Blue MRF′ Ultracompetent cells were transformed. Thetransformed cells were spread onto a 2×YT agar medium containing 50μg/ml ampicillin (Maniatis, Molecular Cloning: A Laboratory Manual, 2nded., Cold Spring Harbor Laboratory Press, New York, 1989), and culturedovernight at 37° C. Single colonies on the culture plate were picked upat random, and cultured overnight at 37° C. with shaking in 2 ml of 2×YTmedium (Maniatis, Molecular Cloning: A Laboratory Manual, 2nd ed., ColdSpring Harbor Laboratory Press, New York, 1989) containing ampicillin ata final concentration of 50 μg/ml. The plasmid was extracted by thealkaline method according to the standard procedure (Muramatsu, LabManual of Genetic Engineering, 3rd ed., Maruzen, Tokyo, 1996, pp.51-55),and dissolved in 50 μl of TE buffer.

The purified plasmid DNA (equivalent to 4 ng of the single-stranded DNA)was alkaline-denatured with 0.5 N NaOH and blotted onto a nylon membrane(No. 1209299, Boehringer Mannheim). The membrane was baked at 120° C.for 30 minutes. Using (CA)₁₀ oligonucleotide digoxigenin-labeled withthe DIG oligonucleotide labeling kit (DIG Oligonucleotide Tailing Kit,Boehringer Mannheim) as a probe, and using the DIG nucleic aciddetection kit (DIG Nucleic Acid Detection Kit, Boehringer Mannheim),(CA)_(n)-positive plasmids were detected according to the manufacturer'sinstruction. Part of the results is shown in FIG. 1.

(5) Cycle Sequencing

For the (CA)_(n)-positive clones, nucleotide sequences were determinedusing the cycle sequencing method. A KS primer labeled with Cy5 at its5′ end, a Reverse primer, and a sequencing kit (ThermoSequencefluorescent labeled primer cycle sequencing kit with 7-deaza-dGPT;Amersham) were used for the sequencing reaction. The fluorescent DNAsequencer (ALFexpress, Pharmacia) was used for sequencing. Based on thenucleotide sequence data thus obtained, optimal primers were designedfor the region encompassing the CA repeats using software for primerdesign (Primer Premier; Premier Biosoft International). The nucleotidesequences of the microsatellite sequences isolated from the blackabalone are shown below. Repetitive units are shown in the parenthesesso that they are easily recognized. The actual sequences are as shown inthe sequence listing (the numbers correspond to the SEQ ID NOs). For theregions 1 to 24, PCR primers can be designed, and a polymorphism wasidentified for nine regions (1, 3, 8, 10, 13, 15, 16, 19, and 24).

-   1. (CA)₄₁-   2. (GACT)₂(CTCA)₇(CA)₂CT(CA)₉-   3. C₅CAC₂(CA)₁₂TA(CA)₈-   4. (CA)₇-   5. (CA)₁₆-   6. (GA)₂CAGA(CA)₅-   7. CA₃GA₂C₃A₃(CA)₅-   8. (CGCA)₉TGCAC₂(CA)₂-   9. (CT)₃(CA₂)₃(CA)₅-   10. (CA)₂₅-   11. CACT(CA)₁₆TACA-   12. CA₃(CA)₂T(CA)₄-   13. (CA)₃₀-   14. CA₂GCA₂C(CA)₂₅-   15. (CA)₂CT(CA)₁₃(CGCA)₁₁(CA)₆-   16. (CA)₈(CG)₄-   17. (CA)₆(CG)₄-   18. (CA)₅-   19. (CA)₂₆-   20. TACATA(CA)₁₂-   21. (CA)₂CA₃(CA)₆-   22. (CA)₂AC(CA)₃AC(CAC)₂(CA)₅-   23. (CA)₈(TGCA)₂-   24. (CA)₃₄-   25. (CA)₇(CGCA)₂CGA₂(CGCA)₂A₂(CA)₂(CG)₂-   26. (CA)₈-   27. CAC₈(CA)₉C₄-   28. (CA)₆(GA)₂-   29. (CA)₂₆-   30. (CA)₂₅-   31. CAG(CA)₅TACA-   32. (CA₃)₃(CA)₄CA₃(CA)₁₂G₂CA(CG₂)₃

About 85% (41 clones) of the (TG/CA)_(n)-concentrated libraryconstructed based on the sequence-independent cleavage method usingsonication was found to be (CA)_(n)-positive clones (FIG. 1). Out ofthem, 32 (CA)_(n)-positive clones were selected at random and sequenced.As a result, PCR primers were designed for 24 clones (72%). For the restof the clones (9 clones), it was impossible to design the primersbecause some repeat regions were adjacent to the ligation site on thevector, because some regions were ligated into the vector in the middleof the repeat, or because some repeats were scattered throughout the DNAfragment.

INDUSTRIAL APPLICABILITY

The present invention makes it possible to efficiently isolate satellitesequences such as microsatellite sequences. Since the present inventionutilizes a sequence-independent cleavage method for fragmentation of agenomic DNA, it is not influenced by the nucleotide sequence, andenables the production of unbiased libraries. Satellite sequencesisolated from the unbiased library contain reduced redundancy. Thus, thepresent invention enables efficient isolation of satellite sequences.

For instance, the present inventors have succeeded in obtaining a(TG/CA)_(n)-concentrated library comprising 32 clones by a singlemanipulation from black abalone, a source from which no microsatellitesequences have been isolated previously. The clones obtained from thislibrary are free from redundancy. It can be concluded that, comparedwith the previous method that produced many duplicated clones forchicken, the present method has increased the efficiency of theisolation. The isolated clones can serve for designing PCR primers aswell as show reduced sequence redundancy, which is another strikingeffect of the present invention. For instance, calculated from theresult of the example, at least 60% (0.85×24/32) of the clones culturedon the plate can be used to design PCR primers. These results revealthat the methods of the present invention for isolating microsatellitesequences are excellent methods that can be widely applied.

Furthermore, the satellite sequences isolated by the methods of thepresent invention can be utilized as DNA markers, such as fingerprintmarkers, pedigree (or lineage) markers, and genetic linkage markers.

1. An isolation method for microsatellite sequences, wherein a genomicDNA is cleaved by an enzymatic cleavage method, the isolation methodcomprising: a) producing randomly cleaved fragments of the genomic DNAusing DNAse I, wherein the fragments have blunt-ends, b) incorporatingthe fragments into appropriate vectors, and c) selecting, from thefragments, fragments comprising microsatellite sequences.